8/6/2019 Preparation of Frozen Tissues for Sectioning

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Preparation of Frozen Tissues for Sectioning

Materials

O.C.T. compound

Cryomold (25mm x 20mm x 5mm)

Dewar or ice bucket to hold liquid nitrogen.

Isopentane (2-Methylbutane)

Small container to hold isopentane

Long handled forceps

ring stand and clamp

Ice bucket for dry ice

Procedure

1. Fill dewar with liquid nitrogen.2. Fill small container with approximately 2 inches of isopentane.

3. Suspend isopentane container from clamp on ring stand and lower the

container into the liquid nitrogen. The base of the container should just touch

the top of the liquid nitrogen and cause it to bubble as it cools the container. If

you insert the container too far into the liquid nitrogen the isopentane will

freeze. If this happens, just remove the container and allow the isopentane to

thaw and then place it back in the liquid nitrogen.

4. Label a cryomold with the identifying data of the tissue. Make sure to use a

marker resistant to organic solvents!

5. Place a small amount of O.C.T. in the bottom of the cryomold.

6. Remove the tissue to be frozen.7. Place the tissue oriented with the side you are interested in at the bottom of the

cryomold.

8. Fill the cryomold with O.C.T. compound. Use caution not to move the tissue

when adding the O.C.T. and be sure to remove any bubbles that form near the

tissue with a forceps as these will cause problems with sectioning (cracking

and fragmentation).

9. Place the cryomold into the isopentane. Use caution not to tip the cryomold

and dislogding the tissue. A little preparation here and ordering a pair of long

handled forceps that can open wide enough to grasp the entire cryomold help

here quite a bit.

10. The O.C.T. will freeze into a solid white color. The tissue should be coveredand not visible as you look down on the mold. Allow approximately 1 minute

for freezing of the entire block.

11. Transfer the tissue to and ice bucket with dry ice and continue with the rest of

the tissues.

12. Place tissues in plastic bag or other airtight container and store at -80oC.

Notes: For some tissues (particularly lung and intestine) it is advantageous to infuse

them with a solution of 50% O.C.T. and 50% PBS prior to placement in the cryomold

to preserve the tissue structure.